The technology of delivering genetic material to cells is currently wide spread and finds many applications. With the technology at hand, it is possible to transfer a wide variety of nucleic acids into a wide variety of different cell types. Some of the more classical technologies include calcium phosphate precipitation, liposome mediated transfer, viral vector mediated transfer, particle bombardment and electroporation.
However, there is no ideal system for the transfer of nucleic acid into cells. All of the transfer systems of the art have their advantages and disadvantages. For instance, the calcium phosphate technology has been shown to be very effective in the transfection of many in vitro growing immortalized cell lines, however, transfer of nucleic acid into many primary cells both in vitro and in vivo has proven to be very difficult. In fact, efficient transfer of nucleic acid in primary cells has only become broadly applicable with the development of systems that use viral elements. Such viral vector systems utilize the very efficient mechanisms that viruses have developed to introduce their genetic information into the target cell. One of the best-studied viral vector systems is the adenovirus vector system.
Gene-transfer vectors derived from adenoviruses (so-called adenoviral vectors) have a number of features that make them particularly useful for gene transfer. 1) the biology of the adenoviruses is characterized in detail, 2) the adenovirus is not associated with severe human pathology, 3) the virus is extremely efficient in introducing its DNA into the host cell, 4) the virus can infect a wide variety of cells and has a broad host-range, 5) the virus can be produced at high virus titers in large quantities, and 6). The virus can be rendered replication defective by deletion of the early-region 1 (E1) of the viral genome (Brody and Crystal 1994).
The adenovirus genome is a linear double-stranded DNA molecule of approximately 36000 base pairs. The adenovirus DNA contains identical Inverted Terminal Repeats (ITR) of approximately 90-140 base pairs with the exact length depending on the serotype. The viral origins of replication are within the ITRs exactly at the genome ends. Most adenoviral vectors currently used in gene therapy have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective. (Levrero et al. 1991). It has been demonstrated extensively that recombinant adenovirus, in particular serotype 5, is suitable for efficient transfer of genes in vivo to the liver, the airway epithelium and solid tumors in animal models and human xenografts in immuno-deficient mice (Bout 1996; Blaese et al. 1995). Thus, preferred methods for in vivo gene transfer into target cells make use of adenoviral vectors as gene delivery vehicles. At present, six different subgroups of human adenoviruses have been proposed which in total encompasses 51 distinct adenovirus serotypes. Besides these human adenoviruses an extensive number of animal adenoviruses have been identified (Ishibashi and Yasue 1984).
A serotype is defined on the basis of its immunological distinctiveness as determined by quantitative neutralization with animal antisera (horse, rabbit). If neutralization shows a certain degree of cross-reaction between two viruses, distinctiveness of serotype is assumed if A) the hemagglutinins are unrelated, as shown by lack of cross-reaction on hemagglutination-inhibition, or B) substantial biophysical/biochemical differences in DNA exist (Francki et al. 1991). The nine serotypes identified last (42-51) were isolated for the first time from HIV-infected patients. (Hierholzer et al. 1988; Schnurr and Dondero 1993; De Jong et al. 1999). For reasons not well understood, most of such immuno-compromised patients shed adenoviruses that were rarely or never isolated from immuno-competent individuals (Hierholzer et al. 1988; Khoo et al. 1995; De Jong et al., 1999). The adenovirus serotype 5 (Ad5), is most widely used for gene therapy purposes. Similar to serotypes 2, 4 and 7, serotype 5 has a natural affiliation towards lung epithelia and other respiratory tissues. In contrast, it is known that, for instance, serotypes 40 and 41 have a natural affiliation towards the gastrointestinal tract. For a detailed overview of the disease association of the different adenovirus serotypes see table 1. In this table there is one deviation from the literature. Sequence analysis and hemagglutination assays using erythrocytes from different species performed in our institute indicated that in contrast to the literature (De Jong et al. 1999) adenovirus 50 proved to be a D group vector whereas adenovirus 51 proved to be a B-group vector. The natural affiliation of a given serotype towards a specific organ can either be due to a difference in the route of infection i.e. make use of different receptor molecules or internalization pathways. However, it can also be due to the fact that a serotype can infect many tissues/organs but it can only replicate in one organ because of the requirement of certain cellular factors for replication and hence clinical disease. At present it is unknown which of the above mentioned mechanisms is responsible for the observed differences in human disease association. However it is known that different adenovirus serotypes can bind to different receptors due to sequence dissimilarity of the capsid proteins i.e. hexon, penton, and fiber protein. It is predominantly fiber that is responsible for the initial attachment and thus determines the host range of a serotype. For instance, it has been shown that adenoviruses of subgroup C such as Ad2 and Ad5 bind to different receptors as compared to adenoviruses from subgroup B such as Ad3 (Defer et al. 1990). Likewise, it was demonstrated that receptor specificity could be altered by exchanging the Ad3 with the Ad5 knob protein, and vice versa (Krasnykh et al. 1996; Stevenson et al. 1995 and 1997).
The initial step for successful infection is binding of adenovirus to a cell surface, a process mediated through fiber protein. The fiber protein has a trimeric structure (Stouten et al. 1992) with different lengths, depending on the virus serotype (Signas et al. 1985; Kidd et al. 1993). Different serotypes have polypeptides with structurally similar N and C termini, but different middle stem regions. N-terminally, the first 30 amino acids are involved in anchoring of the fiber to the penton base (Chroboczek et al. 1995), especially the conserved FNPVYP (SEQ. ID. NO. 1) region in the tail (Arnberg et al. 1997). The C-terminus, or knob, is responsible for initial interaction with the cellular adenovirus receptor. After this initial binding secondary binding between the capsid penton base and cell-surface integrins is proposed to lead to internalization of viral particles in coated pits and endocytosis (Morgan et al. 1969; Svensson and Persson 1984; Varga et al. 1991; Greber et al. 1993; Wickham et al. 1995). Integrins are αβ-heterodimers of which at least 14 α-subunits and 8 β-subunits have been identified (Hynes 1992). The array of integrins expressed in cells is complex and will vary between cell types and cellular environment. Although the knob contains some conserved regions, between serotypes, knob proteins show a high degree of variability, indicating that different adenovirus receptors might exist. For instance, it has been demonstrated that adenoviruses of subgroup C (Ad2, Ad5) and adenoviruses of subgroup B (Ad3) bind to different receptors (Defer et al. 1990). Using baculovirus produced soluble CAR as well as adenovirus serotype 5 knob protein, Roelvink et al. (1998) concluded by using interference studies that all adenovirus serotypes, except serotypes of subgroup B, enter cells via CAR.
Although adenovirus vectors have been used to transfer foreign genetic material into a large variety of cell types, skeletal muscle cells (also referred to as muscle cells) and muscle cell specific precursors thereof (myoblasts) have until the present invention at least in part resisted efficient gene delivery by adenovirus vectors. Until the present invention, the limited capability of adenovirus vectors to deliver a nucleic acid of interest to said (precursor) muscle cell was a problem in gene therapy. Effective gene delivery in said cells is desirable, because in the field of gene therapy there is an interest in skeletal muscle as a target for treatment of many different diseases (reviewed in DiEdwardo et al. 1999). For instance, via direct injection of skeletal muscle with either recombinant viruses (Hartigan-O'Connor and Chamberlain 2000; Akkaraju et al. 1999), naked DNA (Li S. et al. 1999) or liposome complexed DNA, researchers are attempting to treat local muscle disease such as Duchenne muscular dystrophy (DMD). In DMD, proper dystrophin protein expression is impaired due to genetic deletions. However, other structural proteins such as, lamanin-alpha-2-chain, or delta-sarcoglyan cause other types of muscle disease and are therefore also subject of investigation (Vilquin et al. 1999).
Besides muscle diseases, a large number of other human diseases can potentially be treated if highly efficient transduction of skeletal muscle would occur, since skeletal muscle presents a large mass of the human body that is easily accessible. For instance, expression of proteins that are either wrongly expressed, or not expressed at all by a patient suffering from one of many glycogen storage disorders could potentially be treated. Genes known to be non-functional are: for instance Aldolase A, Phosphorylase B-kinase, acid maltase, or ormyophosphorylase (reviewed in DiMauro and Bruno, 1998).
Another example of potential areas in which efficient genetic modification of skeletal muscle could be beneficial are ischemia and peripheral vascular disease because induction of angiogenesis has been shown to be therapeutically beneficial (Isner 2000, Schratzberger et al. 2000). Factors that can induce or inhibit angiogenesis are for instance cardiotropin-1, ang1-7, NOSIII, ATF-BPTI, and vascular endothelial growth factor (Bordet et al. 1999; Rivard et al. 1999). Another example is the genetic modification of the skeletal muscle such that it becomes a “production factory” for therapeutic proteins that can exert their therapeutic effect at sites distant from the skeletal muscle (MacColl et al. 1999). Examples of the latter include growth factors and hormones i.e. EPO, TGF-β, TNF-α, or IL1-IL12 (Ueno et al. 2000; Dalle et al. 1999), blood clotting factors i.e. factor VIII and factor IX (Chao et al. 1999)), neurotropic factors i.e. GDNF or NGF (Mohajeri et al. 1999), or lysosomal lipid degradation enzymes i.e. galactosidase, glucocerebrosidase, ceraminidase. Expression of these proteins in the muscle could be beneficial to treat for instance anemia, blood clotting disorders, or Gaucher disease to name but a few.
Because direct injection of recombinant viruses, naked DNA or liposome complexed DNA has so far not resulted in highly efficient genetic modification of skeletal muscle, alternatives of direct administration approaches are explored. In one such strategy, myoblasts cultured from adult skeletal muscle biopsies are isolated. Myoblasts are subsequently genetically modified and re-infused into the patient, for instance in the skeletal muscle, cardiac muscle or perhaps even in the blood stream giving the fact that, these cells might possess the ability to home to skeletal muscle (Jackson et al. 1999). Transplantation into ischemic regions of a diseased heart of genetically modified myoblasts expressing angiogenic factors might counteract heart failure (Scorsin et al. 2000). This strategy in which genetically modified cells are transplanted directly, is an autologous procedure meaning that cells have to be derived from the patient itself. To broaden the number of applications several polymers have been constructed in which allogeneic cells are encapsulated using semi-permeable membranes which thus shields the cells from the immune system of the host (Li R H. et al. 1998 and 1999). Encapsulated myoblasts thus secrete therapeutic proteins but the myoblasts are protected from the immune system and/or complement inactivation (Regulier et al. 1998; Dalle et al. 1999). Encapsulated cells are surgically implanted at any easy accessible site in the body, i.e. subcutaneous, intra dermal, intra peritoneal, or intra muscular. Another alternative strategy for the direct administration of recombinant viruses, naked DNA, or liposome-complexed DNA is the use of myoblasts for ex vivo tissue engineering also referred to as a “neo-organ approach”. This strategy is based on the implantation of cells, either or not genetically modified, in biodegradable scaffolds in vitro (Rosenthal and Kohler 1997, Yoo and Atala 1997, Moullier et al. 1993). The cells adhere to the scaffold after which the scaffold is surgically transplanted. This approach can be used to generate bioartificial muscle (BAM) in bioreactors (Powell et al. 1999). Genes of interest to transfer to myoblasts using this approach are many and include all examples listed so far. Naturally the, same approaches can be followed when undesired genes or proteins have to be eliminated from the body. Hereto, for instance anti-sense molecules (Soreq and Seidman 2000), ribozymes (Morino et al. 20.00), or chimeraplasts (Rando et al. 2000) can be expressed in myoblasts.
From the above non-limiting, examples it can be concluded that efficient transduction of skeletal muscle cells and myoblasts is of great importance. However, although researchers perform a great variety of beneficial applications with gene therapy involving skeletal muscle cells and myoblasts, until the present invention there were no suitable methods for transduction of a skeletal muscle cell and/or a myoblast, a muscle cell specific precursor of at least a skeletal muscle cell.
Transduction of a (precursor) muscle cell with an adenovirus vector was not efficiently possible. Until the present invention, gene delivery was performed in a variety of ways, which were for instance laborious, and/or inefficient. The present invention solves the problem, how a muscle cell and/or a specific precursor thereof can be efficiently infected by a gene delivery vehicle, preferably comprising an adenoviral vector. With the present invention it is for instance possible to perform a wide range of beneficial applications on a wide scale. An efficient way of transducing said (precursor) muscle cells by an adenovirus vector might for instance improve the time and/or extent of treatment of a disease by gene therapy.